EBV is detected in epithelial cells in a unique oral lesion known as "hairy" leukoplakia (HLP), which is a harbinger for the development of the acquired immunodeficiency syndrome (AIDS). In contrast to most EBV- associated diseases, HLP is a permissive EBV infection with abundant viral replication. We have shown that HLP may harbor both EBV Types 1 and 2 within a lesion and that co-infection with multiple strains of EBV may occur. We have also determined that intertypic recombination and the generation of new EBV variants and defective EBV genomes is a frequent occurrence in HLP and that the strains of EBV which are replicating on the tongue in HLP infect the peripheral blood. These studies have revealed that there are at least seven different strains of EBV that can be distinguished by sequence changes within LMP1. We have also shown that HLP has a distinct pattern of viral expression with concurrent expression of viral genes expressed in permissive. infection and the viral proteins characteristic of latent, transforming infection in lymphocytes, including EBNA2, EBNA-LP, and LMP1. To farther understand the pathogenesis of HLP, the proposed studies will develop in vitro epithelial cell infection systems using laboratory isolates and then use these systems to characterize infection with wild type isolates. Specifically, in Aim l, cultured epithelial cells will be used to determine if there are differences in the degree of infection and viral expression during infection of different epithelial cell lines, between infections with virus produced by lymphoid or epithelial cells, or whether entry is mediated by the known viral receptor, CD21, or by co-cultivation. We will determine if expression of CD21 contributes to superinfection and whether superinfection induces viral replication. In Aim 2, we will utilize our new method of distinguishing strains by LMP1 sequence changes, in addition to EBNA typing, to identify the strains of EBV which are replicating on the tongue in HLP and in peripheral blood lymphocytes. This method and EBNA typing will be used to determine if a subset of these wild type EBV strains can be enriched by passage in epithelial cells in vitro. The wild type isolates will be characterized for their relative ability to infect and establish latent or permissive infections in the cultured epithelial cell systems developed in Aim l. These studies will determine if potentially lytic or epithelial-trophic isolates of EBV are present in HLP and the strains will be captured for biologic and biochemical characterization. To determine the contribution to HLP pathogenesis of the latent, transforming EBV genes LMP1, EBNA2, and EBNA-LP that we have identified in HLP, we will analyze the effects of LMP1, EBNA2, and EBNA-LP on viral infection and epithelial cell growth. In Aim 3, we will determine the effects of LMP1 on viral infection and replication in polarized epithelial cells and raft cultures and determine the molecular properties of the LMP1 variants. In Aim 4, we will determine the effect of EBNA2 and LP on EBV expression early after infection and on expression of CD212 and other cellular genes. Our studies of HLP have revealed that HLP is a novel infection, which we hypothesize is an overt manifestation of the pathobiology of EBV during AIDS. The proposed studies will identify and characterize the viral determinants of disease, further our understanding of oral manifestations of EBV reactivation, and illuminate the complexities of EBV infection in HIV infected individuals.